Fungal infections are increasing as a result of AIDS, transplantation, and high dose chemotherapy. However, existing antifungal agents are limited to amphotericin B, azoles, and flucytosine, and drug resistant isolates are emerging. We propose to elucidate signal transduction cascades regulating virulence of Cryptococcus neoformans, the leading cause of fungal meningitis and a common opportunistic pathogen. Several features make C. neoformans an ideal model fungal pathogen. The organism exists as haploid cells with a defined sexual cycle. Gene disruption by homologous recombination is now possible, and established animal models permit analyses of mutant strains and candidate drugs in a virulence setting. These advances provide tools to elucidate signaling cascades regulating virulence traits of this organism, including capsule and melanin production in response to host signals, and to explore the association between virulence and MATalpha mating type. We have discovered that the G protein Gpa1 is required for mating and capsule and melanin production in C. neoformans. gpa 1 mutant strains are avirulent in an animal mode of cryptococcal meningitis. The phenotypes of gpa 1 mutant cells are suppressed by cAMP, suggesting Gpa1 regulates an adenylyl cyclase/cAMP/protein kinase A cascade. We have cloned C. neoformans homologs of Ras1, the PKA catalytic subunit and the Sch9 kinase, which are known to regulate an analogous G protein/cAMP dependent signaling pathway that regulates pseudohyphal differentiation in S. cerevisiae. In both S. cerevisiae and C. albicans, a MAP kinase signaling pathway also regulates filamentation and virulence. We and others have identified components of a homologous MAP kinase cascade in C. neoformans. Our studies reveal mutants lacking the G beta protein Gpb1, the MAP kinase Cpk1, or the STE12 transcription factor homolog have mating defects and virulence studies with these mutants are in progress. We propose to delineate the MAP kinase and G protein/cAMP dependent signaling cascades regulating C. neoformans virulence to understand regulation of pathogenesis and to identify novel antifungal drug targets.